INFLUENCE OF MARGINAL ADIPOSE TISSUE INVASION IN PROGNOSIS OF DUCTAL CARCINOMA OF THE BREAST AND
ON LYMPH NODE METASTASIS AND ITS CORRELATION WITH HORMONE RECEPTOR STATUS
DISSERTATION SUBMITTED FOR M.D.PATHOLOGY
(BRANCH-III)
THE TAMILNADU DR.M.G.R.MEDICAL UNIVERSITY CHENNAI, TAMILNADU
MAY – 2018
CERTIFICATE FROM THE DEAN
This is to certify that the dissertation entitled “INFLUENCE OF MARGINAL ADIPOSE TISSUE INVASION IN PROGNOSIS OF DUCTAL CARCINOMA OF THE BREAST AND ON LYMPH NODE METASTASIS AND ITS CORRELATION WITH HORMONE RECEPTOR STATUS” submitted by Dr.Rubu Anu to the Faculty of Pathology, The Tamilnadu Dr.M.G.R. Medical University, Chennai in partial fulfilment of the requirement for the reward of M.D. Degree in Pathology is a bonafide work carried out by her during the period 2015-2017.
Place: Madurai Dr.MARUTHUPANDIAN, M.D
Date: DEAN,
Government Rajaji Hospital, Madurai Medical College, Madurai.
CERTIFICATE FROM THE HEAD OF THE DEPARTMENT
This is to certify that the dissertation entitled “INFLUENCE OF MARGINAL ADIPOSE TISSUE INVASION IN PROGNOSIS OF DUCTAL CARCINOMA OF THE BREAST AND ON LYMPH NODE METASTASIS AND ITS CORRELATION WITH HORMONE RECEPTOR STATUS” submitted by Dr.Rubu Anu to the Faculty of Pathology, The Tamilnadu Dr.M.G.R. Medical University, Chennai in partial fulfilment of the requirement for the reward of M.D. Degree in Pathology is a bonafide work carried out by her during the period 2015-2017 under my direct supervision and guidance.
Place: Madurai Dr.T.GEETHA, M.D.,
Date: Professor and HOD,
Department of Pathology, Madurai Medical College, Madurai.
CERTIFICATE FROM THE GUIDE
This is to certify that the dissertation entitled “INFLUENCE OF MARGINAL ADIPOSE TISSUE INVASION IN PROGNOSIS OF DUCTAL CARCINOMA OF THE BREAST AND ON LYMPH NODE METASTASIS AND ITS CORRELATION WITH HORMONE RECEPTOR STATUS” submitted by Dr.Rubu Anu to the Faculty of Pathology, The Tamilnadu Dr.M.G.R. Medical University, Chennai in partial fulfilment of the requirement for the reward of M.D. Degree in Pathology is a bonafide work carried out by her during the period 2015-2017 under my direct supervision and guidance.
Place: Madurai Dr. T.GEETHA , M.D.,
Date: Professor and HOD,
Department of Pathology, Madurai Medical College, Madurai.
DECLARATION BY CANDIDATE
I, Dr.Rubu Anu, solemnly declare that the dissertation titled
“INFLUENCE OF MARGINAL ADIPOSE TISSUE INVASION IN PROGNOSIS OF DUCTAL CARCINOMA OF THE BREAST AND ON LYMPH NODE METASTASIS AND ITS CORRELATION WITH HORMONE RECEPTOR STATUS” is a bonafide work done by me at Department of Pathology, Madurai Medical College & Government Rajaji Hospital, Madurai during the period from July 2015 to August 2017.
I also declare that this bonafide work or a part of this work was not submitted by me or any other for any reward, degree and diploma to any university, board either in India or abroad.
This dissertation is submitted to The Tamilnadu Dr.M.G.R. Medical University, towards partial fulfilment of requirement for the reward of M.D.
Degree in PATHOLOGY.
Place: Madurai. Dr. Rubu Anu
Date:
ACKNOWLEDGEMENT
My profound thanks and gratitude to The Dean, Madurai Medical College and Government Rajaji Hospital, Madurai and the Ethical committee for permitting me to carry out this study.
I wish to express my heartfelt thanks to the respected Professor Dr.T.Geetha, M.D., Professor and Head of the Department of Pathology, Madurai Medical College, Madurai for her valuable suggestions, constant encouragement and guidance throughout this work.
I express my gratitude to all the Professors Dr.G.Meenakumari, M.D., Dr.N.Sharmila Thilagavathy, M.D., Dr.M.Sivakami, M.D., and all the Assistant Professors and Tutors for their valuable suggestions and guidance in this work.
I am grateful to Professor and Head of the department of Surgery and Professor and Head of the department of Surgical Oncology, Government Rajaji Hospital, Madurai for permitting me to carry out this study.
I am indebted to my fellow post graduates and technical staff of the Department of Pathology for their immense help in carrying out this study.
CONTENTS
SERIAL
NO. TITLE PAGE
1 INTRODUCTION 1-2
2 AIMS AND OBJECTIVES 3
3 REVIEW OF LITERATURE 4-46
4 METHODS AND METHODOLOGY 47-51
5 OBSERVATIONS AND RESULTS 52-71
6 DISCUSSION 72-78
7 SUMMARY 79-80
8 CONCLUSION 81
9 ANNEXURES
ANNEXURE I - WHO CLASSIFICATION OF BREAST TUMOURS ANNEXURE II - AMERICAN JOINT COMMITTEE ON CANCER STAGING
ANNEXURE III - MODIFIED BLOOM-RICHARDSON HISTOLOGIC GRADING
ANNEXURE IV - PROFORMA
ANNEXURE V - HEMATOXYLIN AND EOSIN STAINING METHOD ANNEXURE VI - KEY TO MASTER CHART
ANNEXURE VII - MASTER CHART
ANNEXURE VIII - LIST OF ABBREVIATIONS ANNEXURE IX - BIBLIOGRAPHY
ANNEXURE X - ETHICAL COMMITTEE APPROVAL CERTIFICATE ANNEXURE XI - ANTI-PLAGIARISM CERTIFICATE
INTRODUCTION
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INTRODUCTION
Breast carcinoma is a heterogenous disease. It is the most common malignant tumor and the leading cause of carcinoma death in women. It accounts for 23% of all cancers in women globally. Infiltrating ductal carcinoma is the most common type of invasive breast carcinoma (75–80% of all mammary invasive carcinomas).
Various studies have been carried out to assess the prognostic parameters of breast cancer patients. Prognostic factors as such can be categorised into two groups- traditional and molecular. Traditional factors include tumor size, histological grade, vascular invasion, stromal characteristics, lymphatic tumor emboli and angiogenesis. Molecular factors include hormone receptors, p53 and HER2/neu expression.
Functional lymphatics at the tumor margins are responsible for lymphatic metastasis. Tumor cell morphology and proliferation are seen to affect lymph node metastasis. Ability of the tumor to invade the adjacent tissue has been proposed to be an important prognostic factor in many recent articles.
The prognostic significance of adipose tissue invasion at the tumor margins has not been evaluated fully.
Generally, when intraductal carcinoma cells infiltrate the breast stroma, the cells initially penetrate the fibrous tissues,followed by the fibroadipose tissues, and, finally, the adipose tissues in breast cancer . Marginal adipose
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tissue invasion (ATI) may lead to a larger contact area between cancer cells and the peritumoral functional lymphatic endothelium increasing the chances for lymphovascular invasion.
Lymphovascular invasion is a crucial step in the complex process of tumour metastasis. The presence of carcinoma cells in either lymphatic vessels (lymphatic invasion),blood vessels (vascular invasion) or both (lymphovascular invasion) is a significant prognostic factor in invasive breast cancer, with respect to local and distance recurrence and poorer survival . Node-negative patients with lymphovascular invasion had higher breast cancer mortality rate compared with patients with no lymphovascular invasion. The combination of these two microscopic features may be a useful predictive factor in identifying patients who require axillary dissection, chemotherapy, or hormonal treatment.
Present study is intended to evaluate the adipose tissue invasion at tumor margins, lymphovascular invasion and its prognostic significance in carcinoma breast.
AIMS AND OBJECTIVES
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AIMS AND OBJECTIVES
• To determine the influence of adipose tissue invasion of cancer cells at the tumor margins on lymph node status in invasive ductal carcinoma of breast.
• To determine the incidence of lymphovascular invasion in invasive ductal carcinoma of breast.
• To determine the correlation between adipose tissue invasion and lymphovascular invasion.
• To correlate marginal adipose tissue invasion with hormone receptor status.
REVIEW OF LITERATURE
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REVIEW OF LITERATURE
HISTORICAL ASPECT OF BREAST CANCER
• Written records and illustrations of breast cancer date back to 3000-2500 B.C.
• Leonides of Alexandria detailed the approach of incision and cautery.
He stipulated leaving a wide margin of excision and only removing tumours of limited extent, foreshadowing the oncology principles of contemporary surgical practice.
• In the 18th century Cooper’s ligament and Sappey’s subareolar plexus of lymphatics were discovered.
• John Hunter replaced black bile with lymph as the cause of breast cancer.
• Mid years of 19th century saw bold and radical surgeries - The en block resections of Charles Moore in London and Kuster and Volkmann in Germany.
• Axillary node dissections as part of philosophy of extermination were performed in 1882 by William Banks.
• William S Halstead, with its emphasis on removing tissues in one piece to prevent spread and removal of pectoralis major to prevent recurrence then became the undisputed path.
• The hormone dependency of breast cancer was hypothetical, through the observations that disease was aggressive in younger women.
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• Beatson ignited the era of endocrine surgery in 1906.
• Discovery of estrogen in by Jensen in 1967.
• Patey and Handley – modified the radical mastectomy.
• Intraductal carcinoma was defined pathologically early in the twentieth century largely by surgeons interested in the microscopic study of tumors they encountered clinically. Among the first studies are those of J.C. Warren, a surgeon practicing in Boston. Warren's investigation of
“abnormal involution” or cystic disease led him to conclude that carcinoma might develop by transition from hyperplastic duct lesions.
• Cheatle drew heavily upon his own detailed studies of whole-organ sections of the breast to examine the relationships of various lesions to carcinoma as part of a systematic exploration of pathologic processes in the breast.
• Micropapillary carcinoma was illustrated by Cheatle in 1920 and by Bloodgood in 1921, but this term was not used by either author.
• Cheatle referred to the micropapillary proliferation as laciform and noted the cartwheel appearance of carcinoma in a nearby duct. Today, many would describe the cartwheel focus as cribriform. Muir attributed the term cribriform to Schultz-Brauns' article on breast carcinoma contained in Henke and Lubarsch's 1935 Handbook.
• Bloodgood also drew attention to the problem of distinguishing between borderline hyperplastic lesions and intraductal carcinoma.
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• Bloodgood recognized two types of comedo adenocarcinoma, which he referred to as pure comedo adenocarcinoma and comedo adenocarcinoma with areas of fully developed cancer of the breast, the former being entirely intraductal and the latter partly invasive. Follow- up revealed that 30% of node-negative patients with invasive comedo adenocarcinoma developed metastases and died of the disease.
• In 1938 Lewis and Geschickter described 40 patients treated for comedo adenocarcinoma, reporting an 85% 5-year cure rate, with most 5-year survivors having remained well for 10 years
EMBRYOLOGY AND FETAL BREAST DEVELOPMENT
At 5th week of intrauterine life the mammary glands develop from the mammary ridges or milk lines, which are thickenings of the epidermis that first appear on the ventral surface of the fetus extending from the axilla to the upper medial region of the thigh [1] . By 15th week of gestation mesenchymal condensation occurs around the epithelial stalk, the breast bud. Growth of cords of epithelium produces a group of solid epithelial columns which gives rise to lobes in the mammary gland. The papillary layer of the fetal dermis surrounds these growing epithelial cords, and evolves into the vascularized fibrous tissue surrounding individual ducts and their branches which form the lobules.
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Myoepithelial cells arise from basal cells between weeks 23 and 28 of gestation [2] . They play an important role in the branching morphogenesis of the mammary gland by the synthesis of basement membrane constituents such as laminin, type IV collagen, and fibronectin, as well as metalloproteinases and growth factors [3].
The earliest stages of fetal mammary gland formation appear to be independent of steroid hormones, whereas after the 15th week development of the breast structure is influenced largely by testosterone. In the last weeks of gestation, the fetal breast is responsive to maternal and placental steroid hormones and prolactin, which induce secretory activity.
ADOLESCENT BREAST DEVELOPMENT
Adolescent female breast development commences with the onset of cyclical estrogen and progesterone secretion at puberty. Under the influence of estrogen ,ducts grow, elongate and acquire a thickened epithelium [4] . Estrogen dependent differentiation of hormonally responsive periductal stroma also occurs at the same time. Growth hormone and glucocorticoids also contribute to ductal growth. Lobuloalveolar differentiation and growth are enhanced by insulin, progesterone, and growth hormone. The lobules are derived from solid masses of cells that form at the ends of terminal ducts.
ANATOMY OF ADULT BREAST
The average breast measures 10–12 cm in diameter and weighs from 50 gms to more than 400 gms. The adult breast lies between the 2nd and 6th ribs
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in the vertical axis and between the sternal edge and the midaxillary line in the horizontal axis. The breast is attached anteriorly to the dermis by fibrous bands called suspensory (Cooper’s) ligaments, and the posterior to the pectoral fascia.
Approximately 3/4th of the breast lies on the pectoralis major muscle . The breast tissue is divided into upper outer, upper inner, lower outer and lower inner quadrants; the subareolar area; and the axillary tail of the upper outer quadrant.
The arterial blood supply is derived from the axillary, intercostal, and internal mammary arteries,and venous drainage is into the axillary and internal mammary veins. Lymphatic drains to the axillary, subclavicular,and internal mammary lymph nodes.While drainage from the upper outer quadrant is predominantly to the axillary lymph nodes, drainage from the inner quadrants is to the internal mammary chain of nodes. The nerves are branches of the thoracic segmentals.
The nipple is located in the center of the complex surrounded by the areola. Dermis and subcutaneous tissue of the nipple contain smooth muscle bundles arranged radially and longitudinally that serve to identify the nipple histologically whereas areolar dermis has numerous sebaceous glands (the glands of Montgomery) and apocrine glands .The breast consists of 15–20 lobes and each lobe is drained by a collecting duct which connects the nipple with lactiferous sinus. Lactiferous and major ducts connect lactiferous sinus with terminal duct-lobular units (TDLUs). Lobules are composed of terminal ducts and acini and their supporting stroma.The specialized stroma within the
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lobules contains fine collagen fibers, abundant reticulin and numerous small vessels.It is more cellular than the interlobular stroma. Entire duct system is lined by double cell layer of luminal epithelial cells and basally located myoepithelial cells. Depending on the functional status the luminal epithelial cells can be flattened, low cuboidal, or columnar. Myoepithelial cells have ovoid to elongated bipolar nuclei and scanty cytoplasm.
During the early follicular phase of the cycle , normal breast lobules show poorly defined acinar structures with luminal epithelial cells having dark, centrally located nuclei, and eosinophilic cytoplasm. In the luteal phase vacuolization and ballooning of the basally located myoepithelial cells occur due to an increase in glycogen cytoplasmic content. Apical snouts of luminal epithelial cells are present due to the secretory activity.The lumina are enlarged and contain eosinophilic secretory material. Prominent stromal edema is present [5,6,7,8,9] .
IMMUNOPROFILE
The luminal epithelial cells are immunoreactive for low molecular weight cytokeratin such as CK8, CK18, and CK19. These cells also show a heterogeneous reaction for high molecular weight such as CK34BE12 and CK5/6 .
In contrast, majority of myoepithelial cells are negative (or only focally and weakly positive) for LMW-CKs .The myoepithelial cells may show a heterogeneous immunoreaction for HMW-CKs such as CK5/6 or CK34BE12.
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They show positivity to variety of antibodies against actin (smooth muscle actin or muscle-specific actin); smooth muscle myosin,heavy chain; calponin;
S100 protein; p63; CD10; 14-3-3 sigma. Estrogen receptors (ER), progesterone receptors (PR) and androgen receptors (AR) are positive in luminal epithelial cells whereas they are almost always negative in myoepithelial cells [10,11].
INCIDENCE
Breast carcinoma is the most common malignant tumor and the leading cause of carcinoma death in women. It accounts for 23% of all cancers in women globally. Infiltrating ductal carcinoma is the most common type of invasive breast carcinoma (75–80% of all mammary invasive carcinomas) [12].
[ANNEXURE I]
RISK FACTORS
The origin of breast cancer is multifactorial and involves diet, reproductive factors, and hormones. It has been proposed that the common denominator for most of these factors is strong and/or prolonged estrogen stimulation operating on a genetically susceptible background [13] .
Genetic predisposition
Approximately 5–10% of all breast cancers are familial [14] . Germline mutations of two high-penetrance susceptibility genes are associated with a high life-time risk of development of breast cancer as well as some other cancers, in particular ovarian cancer [15]. These are BRCA1, located on
11
chromosome 17q21, and BRCA2, located on chromosome 13q12.3 [15,16].
Mutations of these genes are present in approximately 2% of Ashkenazi Jews.
The risk for breast carcinoma among carriers is estimated to be up to 70–80%
by the age of 70 years[17] .
The BRCA1-encoded protein functions include homologous recombination DNA repair, cell cycle checkpoint control, ubiquitylation, chromatin remodelling, and DNA decatenation. The protein encoded by BRCA2 is involved in DNA repair, cytokinesis, and meiosis [18] . Both BRCA1 and BRCA2 are therefore essential for accurate repair of DNA double-strand breaks by homologous recombination repair.
Analysis of BRCA1 mutations associated breast carcinomas show a higher percentage of tumors with medullary features, i.e., tumors that tend to be of high grade, mitotically very active, with a syncytial growth pattern, pushing margins, confluent necrosis, negativity for hormone receptors and c-erbB-2 (‘triple negative’), basal-like gene expression profile, associated with TP53 mutation. BRCA2-associated cancers on the other hand are a heterogeneous group and commonly positive for hormone receptors. Additional polymorphisms and genes have been identified recently, primarily via genome- wide association studies (GWAS).
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Family history
Women who have a first-degree relative with breast carcinoma have a risk two or three times higher than that of the general population .The risk is further increased if the relative was affected at an early age and/or had bilateral disease [19] .
Menstrual and reproductive history
Early menarche, nulliparity, late age at first birth, and late menopause are associated with increased risk . Breast carcinoma is rare in women who had oophorectomy before 35 years of age as it reduces the risk to one-third. Women who have their first child before the age of 18 years have only one-third the risk of those who had their first child after the age 30 [20] . Reduced risk of breast carcinoma among premenopausal women who have lactated has been documented, but not among postmenopausal women. Risk of breast carcinoma is increased in postmenopausal women with hyperandrogenic plasma hormone profile [20] .
Endogenous hormones
Sex steroids (androgens, estrogens, progestogens) have an important role in the development of breast carcinomas. Incidence of breast-cancer rises more steeply with age before (approximately 8% per year) menopause than after (approximately 2% per year) when ovarian synthesis of estrogen and progesterone ceases and ovarian production of androgens gradually diminishes.
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Among the postmenopausal women , a strong and consistent link between blood concentrations of estrogen and testosterone and risk of developing breast cancer (especially estrogen receptor (ER) positive cancers) is seen. Among premenopausal women, higher follicular concentrations of total and free estradiol and higher concentrations of testosterone are associated with an increased risk of breast cancer but not progesterone or sex hormone- binding globulin (SHBG). Higher concentrations of prolactin are associated with an increase in risk among both pre- and postmenopausal women , more strongly for ER-positive breast cancer [21] .
Contraceptive agents
Various epidemiologic studies that have been conducted in this regard have shown no/ very low risk among young long-term users. However recent studies show current users and recent users (< 10 years since last use) have a modest elevation in risk compared to never-users. IARC has concluded that estrogen-progestogen oral contraceptives (combined) are a class 1 carcinogen[22].
Postmenopausal hormone-replacement therapy
Increased risk of breast carcinoma has been observed in two subgroups – long-duration users and current users . More recently, a large cohort study and a case-control study have provided strong evidence for a greater increase in breast cancer risk in women using hormone replacement therapy than in those using estrogens alone [22,23].
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Adiposity
Case–control and cohort studies have shown an inverse relationship between adiposity (BMI) and incidence of premenopausal breast cancer [24] . Heavier premenopausal women, have more irregular menstrual cycles and increased rates of anovulatory infertility , suggesting that their lower risk may be due to fewer ovulatory cycles and less exposure to ovarian hormones [25] . Amongst postmenopausal women, the association between BMI and risk of breast cancer has been weakly positive .
Fibrocystic disease and epithelial hyperplasia
Increased risk (2- to 9-fold, average 2.5-fold) was observed in patients with a previous diagnosis of fibrocystic disease. Risk of biopsy-proven proliferative breast disease is significantly increased by nulliparity, late age of first birth, and late menopause. These factors are also associated with increased breast carcinoma risk . Dietary breast cancer risk factors such as high intake of meat fat and caffeine have also been associated with a greater risk of proliferative breast disease. Although most of the foregoing investigations did not specify a relationship with particular proliferative changes, the analysis of meat fat revealed a strong association between frequent consumption and an elevated risk for severe atypia and in situ carcinoma . Boyle et al. reported that excess caffeine consumption was associated with atypical lobular hyperplasia and with sclerosing adenosis accompanied by duct hyperplasia. An inverse relationship between dietary fiber content and the risk of benign proliferative
15
breast disease has been reported. A similar relationship was observed between dietary fiber intake and breast density. The mechanism by which dietary fiber influences mammary epithelial proliferation is unknown but it might involve intestinal estrogen metabolism or substances associated with fiber-containing foods such as phytoestrogens. No consistent association between proliferative changes in the breast and a family history of breast carcinoma has been found , although one study reported a slightly higher frequency of atypical hyperplasia in women with a positive family history than among those without . Obesity or excess body mass and the use of oral contraceptives are factors associated with a decreased risk for benign breast disease.
Ionizing radiation
Increased risk of breast carcinoma has been documented with exposure to ionizing radiation, particularly if this exposure occurred at the time of breast development [26]
Others
A peculiar association between breast carcinoma and meningioma has been repeatedly noted. Sometimes even metastasis of the breast carcinoma is found within meningioma[27].
Patients with ataxia–telangiectasia syndrome and with Cowden syndrome have an excess risk of breast cancer [28] .
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LOCATION
Approximately 50% of the tumors are in the upper outer quadrant, 15%
in the upper inner quadrant, 10% in the lower outer quadrant, 5% in the lower inner quadrant, 17% in the central region (within 1 cm of the areola), and 3%
are diffuse (massive or multifocal). The marked difference in the carcinoma frequency depends closely on the amount of breast parenchyma in each quadrant.
Several studies have documented the peculiar fact that breast carcinoma is slightly more frequent in the left breast than in the right. In one recent series, the excess for the left side was 13%.
Multicentricity
Multicentricity (as defined by presence of carcinoma in a breast quadrant other than the one containing the dominant mass) was detected by Fisher et al [29]. Multiple breast carcinoma can result from either intramammary spread of a single lesion or from independent events [30,31] . A recent study showed that multicentric tumours are associated with a lower survival rate than unicentric tumors of the same aggregate volume.
Multicentricity was more common in lobular than in ductal carcinomas.
Bilaterality
The chance that a patient with invasive breast carcinoma will develop a carcinoma in the contralateral breast is about five times that of the general
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population, and is even higher if there is a family history of breast carcinoma.
In cases of lobular carcinoma, the figure can be as high as 25–50% [32,33].
The use of adjuvant chemotherapy significantly decreases the risk of metachronous contralateral breast carcinoma.
METHOD OF TUMOUR DETECTION AND SCREENING
The method of tumor detection influences prognosis and disease-free survival. Among patients diagnosed before widespread mammography screening, tumor detection by clinical breast examination was associated with a significant reduction in recurrence compared with detection by self-palpation.
Screening examinations employing mammography with or without physical examination have been shown to reduce mortality due to breast carcinoma in the screened population.
Tabar et al. demonstrated a reduction in mortality of nearly 30% with mammography screening at 2- and 3-year intervals in Sweden. When compared to breast carcinomas presenting clinically, carcinomas detected by screening with mammography tend to be smaller, to be lower grade, and to have fewer nodal metastases. This difference is most pronounced in the first or prevalence screening examination. The relationship between nodal status and tumor size was similar among patients with carcinomas detected by screening and clinically; the frequency of nodal metastases did not differ significantly according to method of detection within any category of tumor size . Hence,
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the advantage conferred by screening was dependent on detecting smaller tumors.
In one series, 9 (13.6%) of 66 mammographically detected calcifications identified histologically consisted of calcium oxalate crystals, 72.7% were calcium phosphate, and 13.6% were a mixture of calcium oxalate and calcium phosphate . Tornos et al. reported finding calcium oxalate calcifications alone in 2% and in combination with calcium phosphate calcifications in 10.4% of 153 specimens. Calcium oxalate crystals have been responsible for 7.3% and 12% of mammographically localized calcifications that led to biopsy.
Calcium phosphate calcifications and calcium oxalate crystals appear as conventional calcifications in specimen radiographs and in clinical mammograms. Calcium phosphate calcifications typically have high to medium density, and they may have irregular or distinct shapes suggestive of carcinoma in a mammogram, whereas calcium oxalate crystals are likely to appear as polyhedral deposits of low to medium density. An analysis of 2000 screening mammograms revealed that 3% of women examined had two or more polyhedral microcalcifications.
An increase in the number and extent of calcifications raises concern for the presence of carcinoma. Rapid expansion of the area of calcifications has been associated with comedo intraductal carcinoma, whereas slower growth of calcifications characterizes noncomedo intraductal carcinoma.
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The possibility that a noninvasive diagnostic procedure might also detect prognostically significant biologic features of breast carcinoma has been explored with positron emission tomography (PET) using 2-deoxy-2- fluoro[18F]-D-glucose (FDG) to obtain information about glucose metabolism.
Cancer cells exhibit a higher rate of glycolysis than normal cells and they overexpress the immunohistochemically detected glucose transport molecule, GLUT1, which may be responsible for glucose accumulation.
PET has successfully identified breast carcinomas in several studies, and it has been effective as a method for distinguishing between benign and malignant tumors, with a sensitivity of 68% to 94% and specificity of 84% to 97%. Response of breast carcinoma to preoperative chemotherapy has been evaluated by PET.
Oshida et al. studied the differential absorption rate (DAR) of FDG in breast carcinomas by PET, and related the calculated DAR to prognosis in 70 patients after a mean follow-up 41 months. DAR proved to be a significant independent indicator of relapse-free survival in multivariate analysis. DAR was also significantly related to microvessel density in the primary tumors of patients with and without axillary lymph node metastases.
INFILTRATING DUCTAL CARCINOMA (NST)
The microanatomic site of origin of ductal carcinomas appears to be in the terminal duct-lobular unit (TDLU). IDC has irregular or rounded, solid epithelial clusters admixed with single cells and cords of tumour cells lacking
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the morphologic features of any of the special types of invasive carcinoma are present. The carcinoma cells also have a variable appearance. The cytoplasm may be abundant and eosinophilic. Nuclei may be regular and uniform or highly pleomorphic with prominent, often multiple, nucleoli. Mitotic activity may be virtually absent or extensive. Infiltrating and/or pushing margins are seen. The tubules and solid cell clusters are not surrounded by myoepithelial cells. There is no basal lamina around the glands and solid structures. Stromal reaction includes oedematous, myxoid, elastotic changes, or, more often, hypercellular and desmoplastic changes with or without lymphocytic infiltration. Vascular invasion can be identified. Perineural invasion can be present [34].
Invasive duct carcinoma, NST includes tumours that express in part one or more characteristics of the specific types of breast carcinoma, but do not constitute pure examples of the individual tumours. This includes invasive duct carcinomas that have limited microscopic foci of tubular, medullary, papillary, or mucinous differentiation. Slightly more than half of the combined tumours are invasive duct carcinomas with a tubular carcinoma component. If the non- specialized pattern comprises between 10% and 49% of the tumour, the rest being of a recognized special type, then it will fall into one of the mixed groups. The prognosis is likely to be that of the dominant invasive duct carcinoma component.
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Rare morphological variants of invasive carcinoma NST Pleomorphic carcinoma
Pleomorphic carcinoma is a rare variant of high-grade invasive carcinoma NST, which is characterized by a proliferation. of pleomorphic and bizarre, sometimes multinucleated, tumour giant cells comprising > 50% of the tumour cells in a background of adenocarcinoma or adenocarcinoma with metaplastic spindle and squamous differentiation. The tumour giant cells in pleomorphic carcinoma account for > 75% of tumour cells in most cases [35].
The tumours are typically of grade 3 with a high mitotic count and central necrosis. Hormone-receptor expression is usually negative, but may overexpress HER2 protein. Axillary lymph-node metastases are present in 50%
of patients. Many patients present with advanced disease. One recent study found that poor outcome in these tumours is associated with presence of a spindle cell metaplastic component.
Carcinoma with osteoclast-like stromal giant cells
These carcinomas are characterised by the presence of osteoclastic giant cells (OGCs) in the stroma .The giant cells are generally associated with an inflammatory, fibroblastic, hypervascular stroma, with extravasated erythrocytes, lymphocytes and monocytes along with mononucleated and binucleated histiocytes, some containing haemosiderin [36] .
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One third of the reported cases had lymph-node metastases. The 5-year survival rate is around 70%, which is similar to, or better than, patients with ordinary invasive carcinomas. Prognosis is not influenced by the presence of stromal giant cells and is related to the characteristics of the associated carcinoma [37].
Carcinoma with choriocarcinomatous features
60% of invasive carcinomas NST have been found to contain HCG- positive cells and have elevated levels of serum human chorionic gonadotrophin (HCG) [38]. Histological evidence of choriocarcinomatous differentiation, however, is exceptionally rare.
Carcinoma with melanotic features
Combinations of invasive carcinoma NST and malignant melanoma show loss of heterozygosity at the same chromosomal loci in all the components of the tumour, suggesting an origin from the same neoplastic clone .The mere presence of melanin in breast cancer cells is not an evidence of melanocytic differentiation as pigmentation of carcinoma cells with melanin can occur when breast cancers invade the skin and involve the dermoepidermal junction [39,40] .
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STAGING
TNM Staging is based on American Joint Committee on Cancer definition of Primary Tumor (T), Regional Lymph Nodes (N) and Distant Metastasis (M) [41]. [Annexure II]
PROGNOSIS
Breast carcinoma is a heterogeneous disease clinically and pathologically. As noted by Sistrunk and MacCarty almost 70 years ago, “It is impossible to foretell the duration of life of all patients with carcinoma of the breast, because the degree of malignancy varies widely, and persons react differently to the disease”.
Innumerable studies have attempted to assess the prognosis of breast cancer patients on the basis of clinical and pathologic parameters. Conventional histopathological prognostic factors broadly group patients into low, intermediate and high risk categories which are helpful in treatment planning.
College of American Pathologist Consensus Statement 1999 classified the prognostic factors into 3 categories. Category I, factors proven to be of prognostic importance and useful in patient management. It includes TNM staging, histological grade, histological type, mitotic figure counts and hormone receptor status. Category II, factors that have been extensively studied biologically and clinically but whose significance remains to be validated statistically. It includes HER2neu, proliferation markers, lymphatic and vascular channel invasion, and p53. Factors in category III includes DNA
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ploidy analysis, microvessel density, EGFR, TGF-alpha, bcl-2 , pS2, cathepsin D . These are factors whose prognostic value have not been studied sufficiently [42].
Prognosis and management of IDC- NST are similar or slightly worse with 35–50% 10-year survival compared to breast cancer as a whole with around 55% 10-year survival.
Tumour Size
The largest diameter of a mammary carcinoma which represents the gross tumor size is one of the most significant prognostic variables. Survival decreases with increasing tumor size and that there is a rise in the frequency of axillary nodal metastases among node-negative patients considered to be at high risk for recurrence (any size tumor estrogen-receptor negative or estrogen- receptor positive tumor larger than 2 cm). The 6th edition of the American Joint Committee on Cancer (AJCC) refers to tumor size as the diameter of the largest invasive lesion when multiple separate invasive tumors are present. [43]
Roger et al. reported the frequency of axillary nodal involvement in relation to tumor size in a study T1a (0-0.5 cm, 3%); T1b (0.6-1.0 cm, 10%);
(1.1-1.5 cm, 21%); and (1.6-2.0 cm, 35%)[44]. According to Finish Cancer Registry study,for patients with T1a-btumors, the corrected 20-year survival rate was 92% , and for T1c group corrected 20-year survival was 75%[45] .
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Tumor Configuration or Shape
Based on gross tumor configuration majority of invasive duct carcinomas can be described as stellate (spiculated, infiltrative, radial, serrated), circumscribed (rounded, pushing, encapsulated, smooth), or having a mixed contour. Some tumors have indistinct borders. However, carcinomas that appear to have circumscribed margins grossly or mammographically may exhibit an invasive growth pattern microscopically.
Infiltrative tumors tend to be larger and more likely to have axillary lymph node metastases than those with circumscribed margins [46]. Tumors with a stellate configuration in which there is focal necrosis were found to have an especially poor prognosis [47] .
Microscopic Histopathologic Prognostic Factors Histological Grade
Grading of ductal carcinomas is an estimate of differentiation and is limited to the invasive portion of the tumor .
Nuclear grading is the cytologic evaluation of tumor nuclei in comparison with the nuclei of normal mammary ductal epithelial cells. The most widely employed system for nuclear grading, introduced by Black and Speer and Cutler et al is usually reported in terms of three categories: well differentiated, intermediate, and poorly differentiated .[48,49]
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Histologic grading evaluates the microscopic architectural growth pattern of invasive ductal carcinomas as well as cytologic features of differentiation. The most widely used histologic grading systems are based on criteria established by Bloom and Richardson, and Elston and Ellis[50,51]. The parameters measured are: (a) the extent of tubule formation; (b) nuclear hyperchromasia, pleomorphism, and size; and (c) mitotic rate. Each of the three elements is assigned a score on a scale of 1 to 3, and the final grade is determined from the sums of the scores. Histologic grade is traditionally expressed in three categories: scores 3 to 5, well differentiated (grade I); scores 6 to 7, intermediate (grade II); and scores 8 to 9, poorly differentiated (grade III). [ANNEXURE III]
Mitotic rate was reported to be the most important feature of the Bloom- Richardson grading system by Parham et al.[52]. They found that a grading system or prognostic index grade (PIG) based on mitotic rate and the presence or absence of necrosis was a better predictor of outcome than the Bloom- Richardson grading method.
In the original study by Elston and Ellis, using a microscope field with a diameter of 0.59 mm and an area of 0.274 mm2, tumors with a count of 0–9 mitotic figures per 10 hpf were given 1 point, those with 10–19 mitotic figures per 10 hpf scored 2 points, and those with 20 or more mitotic figures per 10 hpf scored 3 points.
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Jannink et al. compared four methods for assessing mitotic activity and concluded that the traditional mitotic activity index (MAI) was preferable because it was easy to apply and less time consuming. In their report, the MAI was measured in a 0.5 ÷ 0.5-cm area in the most cellular region at the periphery of the tumor (selected to avoid areas of necrosis, inflammation, calcification and large vessels. Counting was done at 400x magnification and was limited to invasive tumor in 10 consecutive fields. When determined by a standardized protocol, MAI proved to be a reliable and reproducible method that added significantly to the prognostic value of lymph node status and tumor size. [53] . Nuclear and histologic grades have been shown to be useful predictors of prognosis for patients stratified by stage of disease, especially among those without axillary lymph node metastases. The absence of tubule formation is a particularly unfavorable histologic feature when combined with poorly differentiated nuclear cytology.
Histologic grade has been shown to be significantly related not only to the frequency of recurrence and death due to invasive ductal carcinoma, but also to the disease-free interval and overall length of survival after mastectomy regardless of clinical stage. Increasing tumor grade has been associated with greater tumor size, diagnosis at a relatively young age, and absence of estrogen-receptor expression and an increased risk for breast recurrence after conservation therapy.
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Necrosis
The independent prognostic significance of tumor necrosis has been studied extensively. Gilchrist et al. found that tumor necrosis defined as the
“presence of confluent necrosis of any dimension in a section of invasive cancer that could be distinguished at intermediate magnification,” was a significant predictor of tumor recurrence time and overall survival with 10-year follow-up. However, the effect was manifested only during the first 2 years of follow-up. For patients who remained disease free beyond 10 years, having had necrosis in the primary tumor no longer was a significant prognostic factor [54].
Extensive necrosis is a prognostically unfavorable feature in invasive mammary carcinoma, possibly reflecting a growth rate so rapid that it exceeds tumor sustaining angiogenesis to a substantial degree.
Apoptosis
Apoptosis is an important mechanism of cell death and it may be a factor in tumor necrosis. Apoptotic cells are characterized in routine histologic sections by condensation of chromatin and cytoplasm as well as intra- and extracellular chromatin fragments as small as 2 micrometers. An apoptotic index (AI) can be determined by counting the number of apoptotic cells using the same method as for a mitotic index.
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In one study, high mean AI was associated with poorly differentiated grade, high S-phase fraction and mitotic rate, the absence of hormone receptor expression, and p53 expression [55].
Inflammatory Cell Infiltrate
Stromal inflammatory cells within and around invasive duct carcinomas consists mainly of mature lymphocytes with a variable admixture of plasma cell, histiocytes, neutrophils, and mast cells. Tumors with plasma cell predominance are usually medullary carcinomas or carcinomas with medullary features. Infiltrating duct carcinoma with medullary features, may have a slightly more favorable prognosis than infiltrating duct carcinomas generally [56].
Some investigators have found carcinomas with a “host response” to have a relatively favorable prognosis [57].
Lymphovascular Invasion (LVI)
Lymphovascular invasion refers to the invasion of lymphatic spaces, blood vessels, or both in the peritumoral area by tumor emboli. LVI is a key step of tumor cells reaching lymph node, therefore, LVI has been known as an independent predictor of lymph node metastases, disease free survival (DFS) as well as overall survival (OS).
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Features used to document the presence of lymphatic tumor emboli are the following: (1) the occurrence of the area outside the margin of the carcinoma, (2) the tumor emboli do not conform exactly to the space in which they lie, (3) the presence of an endothelial cell lining, and (4) the presence of blood vessels in the immediate vicinity. If doubts persist, a stain with D2-40 and podoplanin have been shown to be the most sensitive and specific markers for lymph vessel endothelium. Monoclonal and polyclonal antibodies directed at the endothelial hyaluronan receptor-1 (LYVE-1) have also proved to be useful for detecting lymphatic tumor emboli associated with breast carcinomas [58]. CD31, CD34, FLI-1, Ulex europaeus I lectin and Factor VIII-related antigen or Von Willebrand Factor, are selective for blood vessels; staining of lymphatic channels by these markers has been either weak or negative.
The presence of axillary nodal metastases was associated with peritumoral but not with intratumor lymphatic invasion. When studied in H&E sections alone, peritumoral lymphatic tumor emboli are found associated with approximately 15% of invasive duct carcinomas. The majority of these patients have axillary lymph node metastases, but lymphatic tumor emboli are found in the breast surrounding invasive duct carcinomas in 5% to 10% of patients who have negative lymph nodes in routine H&E slides. Several studies have shown that peritumoral lymphatic emboli were prognostically unfavorable in node- negative patients treated by mastectomy and by breast conservation therapy.
Definite invasion of the lymph vessels, and blood vessels was found by Fisher et al. in 33%, and 5% of cases, respectively[59].
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At the St. Gallen meeting in 2005, lymphovascular invasion was recognised as a prognostic factor for node negative patients. Node-negative patients with lymphovascular invasion had higher breast cancer mortality rate (53%) compared with patients with no lymphovascular invasion (29%).The association seems to be stronger if the lymph vessel nature of the involved spaces is confirmed by performing immunostaining for the endothelial cell marker D2-40.
Lymph vessel invasion may be difficult to distinguish from artifactual tissue retraction. However, Acs et al. found that there was a significant direct correlation between the presence of shrinkage artifact and the presence of lymphatic tumour emboli in node-negative patients. Furthermore, node- negative patients with shrinkage artifact had a significantly higher frequency of distant metastases than patients without shrinkage artifact. These findings led the investigators to suggest that shrinkage artifact may reflect significant aspects of tumour-stromal interaction, possibly related to the formation of lymphatic channels, and not simply a passive phenomenon due to incomplete tissue fixation [60].
Axillary Node Status
Nodal status is the most powerful independent prognostic factor in breast cancer and remains the most important feature for defining risk category.
The first route of spread for breast carcinoma is through the axillary lymph nodes, and the incidence of ALN metastasis increases with larger tumours.
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Even patients with T1a and T1b disease have significant nodal involvement (5- 15%). There is evidence that overall survival decreases as the number of positive node increases.
When lymph nodes are affected, metastases are found in a stepwise fashion in the low, mid-, and upper or proximal axillary zones. Several authors reported that discontinuous or “skip” metastases, which do not follow this distribution, were found in 2% or less of all patients or in less than 5% of patients with axillary nodal metastasis. Metastases limited to level II (mid- axilla) account for 30% to 50% of the instances of discontinuous involvement.
In a study of more than 1000 patients with a mean follow-up of 97 months, who underwent complete axillary dissection (levels I, II, and III), tumour size, the number of involved lymph nodes, and the level of involvement were independent predictive factors for survival . Careful manual dissection of the unfixed axillary fat is the most cost-effective method for isolating lymph nodes for microscopic study.
Intraoperative mapping of the lymphatic drainage from the breast has proved to be an effective method for locating one or more lymph nodes most likely to harbor metastatic carcinoma, called Sentinel Lymph Node (SLN). An important advantage of SLN mapping when compared with conventional axillary dissection is reduced postoperative morbidity. Patients experience significantly less lymphedema and other symptoms after SLN mapping than after axillary dissection. The procedures now widely employed involve injection of a vital blue dye, a radioactive tracer such as technetium-99m-sulfur
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colloid, or a combination of these reagents. Blue dye transported to the SLN can be identified visually.
The distinction between heterotopic mammary glands and metastatic carcinoma is usually not difficult. The glandular structures typically occur outside of or within the lymph node capsule or in the lymphoid tissue rather than in nodal sinusoids. Myoepithelial cells and specialized intralobular stroma are evident in some heterotopic mammary lobules associated with lymph nodes. In the absence of myoepithelial cells, glands in axillary lymph nodes usually represented metastatic carcinoma, even if the growth pattern is well- differentiated. These metastatic foci are typically distributed as isolated glands or small groups of glands in the lymphoid tissue of the lymph node, or in the capsule and subcapsular lymphatic spaces. Rare instances of very orderly glands lacking demonstrable myoepithelium in axillary SLNs were demonstrated for which no corresponding carcinoma could be detected in the ipsilateral breast. These might be an unusual form of benign glandular inclusions or the result of displacement of benign epithelium from a prior biopsy site in the breast. The latter phenomenon has sometimes been referred to as “benign transport.” Metastatic carcinoma derived from an occult primary breast carcinoma can only be excluded in this circumstance if a mastectomy is performed and the specimen is thoroughly studied.
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According to St. Gallen experts, involvement of four or more nodes in the axilla by itself indicated high-risk, but patients with one to three nodes involved required HER2/neu overexpression or amplification to be included in the high-risk group, while other patients with one to three nodes included in the intermediate-risk category.
Although nodal micrometastases were prognostically relevant in several studies, neither they nor isolated tumor cells in lymph nodes are considered in risk allocation. Study by Yenidunya et al. Showed that a significant proportion of patients diagnosed as axillary node negative (ANN) using standard histopathological techniques may have occult nodal metastases (OMs). OMs were more frequently found in lobular (38%) than ductal carcinoma (25%), and more frequently in women less than 50 years (41%) than in older women (19%). The presence, size and number of OM had prognostic significance just as tumour size and histological and nuclear grade [61].
Marginal Adipose Tissue Invasion
Yamaguchi et al defined Marginal ATI as the presence of more than 20 cancer cells in direct contact with the adipose tissue or the location of cancer cells in the adipose tissue and LVI was defined as the presence of peritumoral lymphatic emboli in endothelium-lined spaces.
The breast stroma histologically consists of fibrous tissues and adipocytes in variable proportions, and the adipocytes physiologically increase with aging in older women. Generally, when intraductal carcinoma cells
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infiltrate the breast stroma, the cells initially penetrate the fibrous tissue, followed by the fibroadipose tissues, and, finally, the adipose tissues (ATI) in breast cancer. The study found ATI to be an independent factor influencing nodal metastasis. Patients with ATI had a poorer prognosis than patients without ATI (10-year disease free survival, 76% and 94%, respectively). In addition, patients without ATI or LVI had neither lymph node metastasis nor recurrent disease. The study found ATI as one of the biologic indicators of tumour aggressiveness [62].
I. Kimijima et al described the following criteria to indicate fat invasion (FI) or scatter invasion of fats (SIF) positivity: the tumor margin faces surrounding fat and is shown in more than 10% of the tumor circumference.
Concluding SIF indicates that cancer cell clusters have spread into surrounding fat in a scattered manner without a dominant anchor of fibrous tissue around them. Each cluster must consist of about 50 or fewer cancer cells. If this finding was shown in 10% or more of the tumor circumference, it was diagnosed as SIF positive [63].
Breast cancer cell growth is estrogen-dependent and the ovary is the main estrogen source in premenopausal women. Cancer cells proliferate under the conditions of very low estrogen levels in postmenopausal women. Recently, the role of intratumoral environment or the intracrine system for cancer growth is better understood.
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Studies have found that even in postmenopausal women, estrogen levels in breast cancer tissue are high enough for estrogen-dependent growth and the estrogen source is considered to be mainly surrounding adipose tissue that contains aromatase which converts androgen to estrogen. This hypothesis was proved by the fact that neoadjuvant therapy with aromatase inhibitor could reduce the intratumoral level of aromatase activity and estrogen. In premenopausal women, fibrous or collagenous tissue is dominant. A close relation between ER status and FI or SIF status only in postmenopausal patients has been confirmed.
In 2000 Hasebe et al proposed a new classification scheme for IDC to predict the prognostic outcome of the IDC patients. This nucleus-fibrotic- focus-fat invasion classification was based on nuclear atypia, fibrotic focus and invasive length of fat invasion (ILFI ). A score of 1each was given to nuclear atypia 3, fibrotic foci and ILFI >2mm. Absence of any of these factors was given a score 0. A significant high hazard ratio was observed in IDC with NFF score 3.
Sasano H et al found that aromatase activity and expression is most prominent proximal to tumour in breast adipose tissue. Aromatisation of in situ estrogen production by aromatase has been considered to play an important role. Aromatase activity and adipocytokines such as leptin present in the breast adipose tissue may have a role in ATI associated nodal involvement.
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Angiogenesis
The acquisition of the angiogenesis is attributed to the overexpression of angiogenic factors, such as vascular endothelial growth factor (VEGF), also termed vascular permeability factor (VPF) and basic fibroblast growth factor (bFGF)[64]. Mammary stromal fibroblasts produce VEGF and the expression of this protein is upregulated by exposure of the cells to hypoxic conditions.
Angiogenic proteins can be produced by inflammatory cells present in the stroma such as macrophages. Expression of VEGF protein and mRNA were higher in invasive ductal than in invasive lobular carcinomas studied by Lee et al [65].
Tumor growth is enhanced not only by increased perfusion associated with neovascularization, but also by the paracrine mitogenic effects of growth factors, such as insulin-like growth factor 1 (IGF-1) and platelet-derived growth factor (PDGF) produced by endothelial cells .
Current attempts to interfere with the VEGF pathway have concentrated on the development of monoclonal antibodies to VEGF or VEGF-receptor (VEGFR) and on tyrosine kinase inhibitors. Bevacizumab is a humanized recombinant antibody to VEGF, which is the first of these agents to be approved for clinical trials in combination with chemotherapy [66].
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Perineural Invasion
Perineural invasion can be found in approximately 10% of invasive carcinomas. It tends to occur in high-grade tumours, frequently associated with lymphatic tumour emboli, but it has not been proven to have independent prognostic significance.
Stromal Characteristics
There is a strong association between stromal characteristics and prognostically significant structural features of breast carcinomas. Tumors that contain minimal stromal reaction tend to have the following characteristics:
circumscription, poorly differentiated nuclear and histologic grade, and a prominent lymphoplasmacytic reaction and are estrogen-receptor negative. On the other hand, densely fibrotic or scirrhous carcinomas are often stellate, moderately differentiated, and have little lymphoplasmacytic reaction. These lesions are estrogen-receptor positive in most of the cases.
Invasive ductal carcinoma of the breast with fibrotic focus was first described in the pathology literature in 1996 by Hasebe et al. [67].
Histologically, a fibrotic focus is a mixture of fibroblasts and collagen fibers that can occupy varying amounts of the volume of an invasive ductal carcinoma. The fibrous bands expand radially into the surrounding tumour and resemble the appearance of a scar. The fibrotic focus is always surrounded by a highly cellular zone of infiltrating ductal carcinoma cells that occupy varying percentages of the tumour area.
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This feature has been shown to be an indicator of tumour aggressiveness, associated with a significantly poorer survival than that seen in invasive ductal carcinoma without a fibrotic focus, with a predilection for brain and lung metastasis. These lesions exhibit higher tumour aggressiveness and proliferative activity, overexpression of c-erbB-2 protein, nuclear accumulation of p53, high proliferative cell nuclear antigen index, aneuploidy, and high tumour angiogenesis.
Colpaert and colleagues subsequently showed fibrotic focus to be a marker of intratumoral hypoxia that promotes angiogenesis, thereby rendering fibrotic focus a marker for increased angiogenesis. This may, in turn, allow tumours to reach their full metastatic potential [68].
Hormone Receptor Status
Estrogen Receptor (ERs) and Progesterone Receptor (PRs) bind hormones that exert their effects in the nucleus. Nuclear immunostaining for both receptor proteins can be demonstrated in normal breast acini, which serve as internal controls for the testing procedure. Nuclear staining in normal breast tissue is heterogeneous and varies with the menstrual cycle. One effect of estrogen is to induce the progesterone; thus the coordinate expression of both hormones in the same cell reflects the intactness of the ER/PR axis in the cell.
In carcinomas of the breast, most PR-positive tumours are also ER positive. ER negative, PR-positive tumors account for <5% of all breast cancers. Approximately 70–80% of invasive breast carcinomas NST are ER-
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positive and approximately 15% of cases are HER2-positive. The overall co- expression of hormones receptors in the study by Colomer et al., reported ER+/PR+, ER+/PR–, ER–/PR+, and ER–/PR– in 46%, 19%, 7% and 28%, respectively .
Since the early 1990s, the IHC assay determination of ER/PR levels has replaced the dextran-coated charcoal (DCC) method, also called the ligand binding assay (LBA).
The effect of quantitation and establishing that ER is a continuous variable was clearly shown by Harvey and coworkers in 1999 using the Allred score in their series of 1982 primary breast cancer cases [69]. An Allred score of 0, 2, 3, 4,5, 6, 7, and 8 was seen in 26%, 3%, 6%, 10%, 16%,19%, 16%, and 4% cases, respectively. The authors demonstrated that there was a linear correlation between Allred score and ER content, as measured by LBA. This study also showed differences in disease-free survival based on the Allred score. Even the current reverse transcriptase polymerase chain reaction (RT- PCR) assay for HR has shown a broad dynamic range of HRs that are present in tumor cells.
The ERBB2 (HER2) gene was originally called NEU as it was first derived from rat neuro/glioblastoma cell lines. Coussens and coworkers named it HER2 because its primary sequence was very similar to human epidermal growth factor receptor (EGFR or ERBB or ERBB1) [70]. Approximately 15%
to 20% of breast cancers demonstrate HER2 gene amplification or protein
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overexpression. In the absence of adjuvant systemic therapy, HER2-positive breast cancer patients have a worse prognosis (i.e., a higher rate of recurrence and mortality), which clearly demonstrates the prognostic significance of this gene.
HER2 positivity is predictive of response to anthracycline- and taxane- based therapy, HER2-positive tumors generally show relative resistance to all endocrine therapies; however, this effect may be more toward selective endocrine receptor modulators such as tamoxifen and less likely toward estrogen depletion therapies such as aromatase inhibitors.
Trastuzumab is a humanized monoclonal antibody to HER2 that was approved by the FDA in 1998 for use in metastatic breast cancer. Trastuzumab improves response rates, time to progression, and survival when used alone or in combination with chemotherapy in the treatment of metastatic breast cancer.
There is an inverse association between hormones receptors and HER2 expression which leads to lower or absent hormone receptors in women with HER2 positive breast cancers. This is one of the reasons why women who over-express HER2 may be resistant to Tamoxifen.
Novel Molecular Markers
Determination of S-phase Fraction and Ploidy by Flow Cytometry
Atkin and Kay described the relationship of modal DNA values to prognosis in 1465 diverse malignant tumors, including some breast carcinomas.
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The study was based on microspectrophotometric DNA measurements made of Feulgen-stained imprints of fresh tumors. Most tumors exhibited a bimodal distribution of DNA values. Generally, patients whose tumors were in the near- diploid range had a better survival than those with an aneuploid DNA distribution. Tumors with a near-diploid DNA distribution tend to be estrogen and progesterone-receptor positive, whereas receptor negativity has been associated with aneuploidy[71].
Pathological study showed correlation between cytologic grading of tumor nuclei and DNA ploidy analyzed by flow cytometry. Low-grade tumors typically exhibit near-diploid DNA, whereas aneuploidy is most pronounced in tumors with high-grade nuclei. Subtypes of carcinoma that are histologically low grade, such as tubular, mucinous, and papillary carcinoma, are usually diploid, whereas medullary carcinoma, which is a cytologically high-grade neoplasm, is generally aneuploid.
The proliferative proportion of cells in DNA synthesis determined by the thymidine labeling index (TLI) has been shown to correlate significantly with prognosis. High TLI has been associated with a higher frequency of recurrence, earlier recurrence, and shorter survival after recurrence.
Flow cytometry provides a method of determining the proliferative fraction (S-phase fraction, or SPF), which is reported to be equivalent to the TLI. In 1983, Hedley et al. described a method of preparing paraffin embedded tissues for flow-cytometry DNA analysis. DNA histograms of cells from fixed
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material correlate well with suspensions of unfixed cells from the same tumor.
Proliferative activity reflected in the SPF is correlated with ploidy to the extent that diploid carcinomas tend to have a lower SPF than aneuploid lesions.
Tumors with a high SPF tend to be estrogen-receptor negative. SPF has been found to correlate with the histologic differentiation of duct carcinomas and with nuclear differentiation[72].
Immunohistochemical Assessment of Proliferation
5-Bromodeoxyuridine (BrdU), a thymidine analog is incorporated into DNA during the S-phase of the cell cycle. Uptake of BrdU after in vivo administration to patients or in vitro incubation of fresh biopsies with BrdU can be measured in tissue sections by an immunohistochemical procedure that employs an anti-BrdU antibody, or by multiparameter flow cytometry. Weidner et al. found that in vivo BrdU labeling was significantly correlated with histologic grade and with the mitotic count [73].
Ki67 is a mouse monoclonal antibody to nuclear components of a cell line derived from Hodgkin's lymphoma [74]. The antibody reacts with a nuclear antigen expressed in proliferating cells throughout the cell cycle but which is absent from quiescent cells . It has been shown that there is a close correlation in breast carcinomas between the Ki67 growth fraction and SPF determined by flow cytometry, the TLI, the BrdU index, labeling with MIB1, and mitotic counts. Because Ki67 labeling occurs throughout the cell cycle, the
